The long-term goal of this project is to understand how ribosomes are assembled in eukaryotes. We use the yeast Saccharomyces cerevisiae for our studies to facilitate combined genetic, molecular biological, biochemical, proteomic, and computational approaches. Ribosome assembly occurs by association of ribosomal proteins with nascent rRNA in the nucleolus, followed by further maturation of pre-ribosomes in the nucleoplasm and cytoplasm. In concert with entry of ribosomal proteins into assembling ribosomes, the rRNA primary transcript undergoes nucleolytic processing to produce mature 18S, 5.8S, and 25S rRNAs. Trans-acting factors associate with these pre-ribosomes, carry out yet to be determined essential "assembly and rRNA processing" functions, then dissociate before mature ribosomes begin to function. These trans-acting factors were identified by screens for yeast mutants defective in ribosome assembly, and more recently by development of methods to purify ribosome assembly intermediates and identify their protein constituents. One specific goal is to combine genetic approaches with the newly developed capabilities to purify and assay assembly intermediates, to define functions of trans-acting factors. How are the dynamic protein, protein-RNA, and RNA-RNA interactions established, disrupted, and reconfigured to establish the final structure of ribosomes? Among the factors are many putative scaffolding proteins, which might nucleate and regulate such molecular rearrangements. We will focus on one of these, Ytm1p, as a prototype to understand scaffolding protein function. Despite the large catalog of trans-acting factors, we know little about interactions among trans-acting factors, ribosomal proteins, and pre-rRNA during assembly. Which proteins contact rRNA? Which proteins interact with each other? Where are these molecules located in assembling ribosomes? Can one define assembly neighborhoods or subcomplexes, as observed for prokaryotic ribosomes assembling in vitro? In what order do these factors associate with, then dissociate from assembly intermediates? By what means are assembly factors and r proteins recruited to and released from pre-ribosomes? Experiments are proposed to address these questions.